PROJECT SUMMARY Genes and their products (RNA and protein) are not expected to intermingle, except in the situation of cancer. However, this traditional dogma is being challenged when more and more chimeric RNAs being identified in non-cancer samples. Our work on trans-splicing and cis-splicing between adjacent genes (cis-SAGe) have provided evidence that the intergenic splicings may be another mechanism to expand functional genome. In this proposal, we are focusing on the genome-wide study of the transcriptional read-through fusions (we named them cis-SAGe to differentiate from other mechanisms). Traditionally, they were believed to be rare, or artifacts. Even when proven true, they were thought to be transcriptional noise or side products. However, in our preliminary studies, we have identified thousands of such fusion RNAs in non-cancer tissues and cells. Some have been validated, and proven functional. We hypothesize that cis-SAGe chimeric fusion RNAs are a widely spread phenomenon in normal physiology, and represent a means to diversify our transcriptome. To test this hypothesis, and to gain knowledge about these fusions at the genome level, we propose the following four aims: In Aim1, we will characterize the fusion RNAs and parental genes including junction sequence, protein-coding potential, expression, and gene ontology. The fusion RNAs will also be analyzed at multiple levels with multiple techniques including non RT-based Nanostring platform, and high throughput mass spectrometry. In Aim2, we will investigate the biological significance of the cis-SAGe fusions. A high throughput screening will be used followed by candidate approaches with both gain- and loss-of-function systems. For the loss-of-function system, we will use RNAi method, which is more mature in our hands. In addition, we will use our newly adapted dCAS9-KRAB method to achieve fusion-specific silencing. We will apply the perturbation in both cell culture and animal systems. One of the fusions will be examined in more detail in a ?retrogenic? mice model. In Aim3, we will investigate the generating mechanisms of cis-SAGe with two approaches, a bioinformatics approach and a reporter approach. Bioinformatically, we will interrogate multi-omics data from ENCODE and Roadmap Epigenomics to investigate epigenetic signatures, transcriptional regulatory patterns, DNA methylation as well as the three-dimensional proximity of the cis-SAGe fusion sites. In the reporter system, we have built a two-part renilla luciferase separated by introns, exons and termination site of a model fusion RNA, and we are using the system to screen candidate factors. In Aim4, we will develop an interactive web-based database to allow end users to search for fusion RNAs in normal tissues and cells. The findings from the proposed study will have the potential of not only challenging traditional dogmas that chimeric RNAs are cancer-specific features, but also enhancing our understanding of the human genome and transcriptome. The chimeric RNAs may be misregulated in disease situations, thus expanding the repertoire for biomarker and therapeutic target discovery.